Cationic dextran polymer derivatives

ABSTRACT

Cationic dextran polymer derivatives including an ester-linked amine-containing substituent and an alkyl ester substituent and methods for making such dextran polymer derivatives are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US2010/056515, filedNov. 12, 2010, which claims the benefit of U.S. Provisional PatentApplication No. 61/261,120, filed Nov. 13, 2009, each of which isincorporated herein in its entirety by reference.

TECHNICAL FIELD

Embodiments of dextran polymer derivatives comprising an ester-linkedamine-containing substituent and an alkyl ester substituent aredisclosed.

BACKGROUND

Pharmaceutically active agents are generally formulated as solid orliquid dosage forms for administration. Such dosage forms generallycomprise the active agent combined with excipients to form materialsthat may be conveniently and reliably administered to a patient in needof such therapy, and following administration, the active agent isabsorbed and distributed in the patient in a way that leads to goodefficacy and safety.

Cationic polymers have previously been used for extended or controlledrelease of active agents near the site of delivery. Some cationicpolymers have been evaluated for this purpose and in some cases havebeen referred to as “mucoadhesive.” Examples of cationic mucoadhesiveexcipients include chitin, chitosan, and amino-substituted polyacrylatesand polymethacrylates.

Dextrans having an amine substituent that possesses at least two aminogroups and a hydrophobic group have been described. The polymers areused in compositions for gene therapy.

Compositions that consist of complexes of copolymers and adenovirusesthat are not bound by covalent bonds and have been used to improvedelivery and transgenic expression of the adenovirus in cells have beendescribed. The copolymers consist of a cationic polymer (such aspolyethyleneimine [PEI], polylysine, diethylaminoethyl [DEAE] dextran,and derivatives) and a nonionic polymer (such as polyethylene glycol[PEG] and derivatives).

Amino-acid substituted dextrans useful for systemic delivery oftherapeutic agents have been described. Amino-dextrans for use informing colloidal particles have also been described.

What is desired is a cationic dextran derivative that would be suitablefor use in a wide variety of formulations.

SUMMARY

In one aspect, a cationic dextran polymer derivate is disclosed. Thedextran polymer derivative comprises (a) an ester-linkedamine-containing substituent, selected from

and mixtures thereof, wherein R₁ is selected from C₁, C₂, C₃, or C₄alkyl groups, R₂ and R₃ may be the same or different and are selectedfrom hydrogen, methyl and ethyl groups, and R₄, R₅, and R₆ may be thesame or different and are selected from methyl and ethyl groups, andwherein the degree of substitution of said ester-linked amine-containingsubstituent is at least 0.03; and (b) an alkyl ester substituentselected from acetate, propionate, butyrate, isobutyrate, and mixturesthereof, wherein the degree of substitution of said alkyl estersubstituent is at least 0.05.

In one embodiment, the ester-linked amine-containing substituent is

wherein R₁ is selected from C₁, C₂, C₃, or C₄ alkyl groups, and R₂ andR₃ may be the same or different and are selected from hydrogen, methyland ethyl groups. In one embodiment, R₁ is a C₂ alkyl group, and R₂ andR₃ are hydrogens.

In another embodiment, the ester-linked amine-containing substituent is

wherein R₁ is selected from C₁, C₂, C₃, or C₄ alkyl groups, and R₄, R₅,and R₆ may be the same or different and are selected from methyl andethyl groups. In one embodiment, R₁ is a C₃ alkyl group, and R₄, R₅, andR₆ are methyl groups.

In one embodiment, the ester-linked amine-containing substituent has adegree of substitution of at least 0.05. In still another embodiment,the ester-linked amine-containing substituent has a degree ofsubstitution of at least 0.1.

In another embodiment, the degree of substitution of the alkyl estersubstituent is at least 0.1. In another embodiment, the degree ofsubstitution of the alkyl ester substituent is at least 0.5. In stillanother embodiment, the degree of substitution of the alkyl estersubstituent is at least 1.0.

The cationic dextran polymer derivatives disclosed herein have uniqueproperties that make them suitable for a wide variety of applications.

By combining an ester-linked amine-containing substituent with an alkylester substituent, compositions containing the cationic dextranderivative can associate with anionic active agents, other anionicformulation materials, and anionic endogenous materials. Such materialscan, among other uses, help retain active agents near the site ofdelivery and action. Specifically, the cationic groups of the cationicdextran polymer derivative can associate with anionic groups on anionicmaterials such as proteins, peptides, and oligoneucleotides. However,the addition of the alkyl ester groups reduces the water solubility ofthe cationic dextran and promotes association with the anionicmaterials. Such association can, in some cases, form complexes,aggregates, nanoparticles, or precipitates.

In addition, the combination of an ester-linked amine-containingsubstituent with an alkyl ester substituent provides tunability andflexibility to the researcher to achieve a polymer with the propertiesthat are ideal for the specific therapeutic target. By adjusting theratio of the ester-linked amine-containing substituent to the alkylester substituent, factors such as aqueous solubility, dissociationconstant (pKa), mucoadhesion, and/or solubility in organic solvents canbe optimized for the required task.

Unlike other cationic polymers, such as amine-functionalized acrylatesor methacrylates (for example, some grades of excipients sold under thename EUDRAGIT®), certain embodiments of the cationic dextran polymerderivatives are biocompatible and biodegradable, and therefore are moresuitable for parenteral administration.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the ¹³C nuclear magnetic resonance (NMR) spectrum ofPolymer 1.

FIG. 2 shows the ¹³C nuclear magnetic resonance (NMR) spectrum ofPolymer 2.

FIG. 3 shows the ¹³C nuclear magnetic resonance (NMR) spectrum ofPolymer 3.

FIG. 4 shows the ¹³C nuclear magnetic resonance (NMR) spectrum ofPolymer 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present disclosure relates to cationic dextran polymer derivativescomprising an ester-linked amine-containing substituent and an alkylester substituent. Embodiments of cationic dextran polymer derivativesand methods for making them are described in detail below.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, percentages, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Cationic Dextran Polymer Derivatives

Dextran polymer derivatives are polymers formed by the derivatization ofdextran with ester-linked groups. Dextran is an α-D-1,6-glucose-linkedglucan. It may have side-chains linked to the backbone of the dextranpolymer, with the degree of branching approximately 5%, and the branchesare mostly 1-2 glucose units long. A representative structure of therepeat unit of dextran is illustrated below.

In one embodiment, the dextran used to form the dextran polymerderivative has a molecular weight that may range from 1,000 to 200,000daltons. As used herein, by “molecular weight” is meant thenumber-average molecular weight as determined by chromatographic methodswell known in the art. In these methods, the number-average molecularweight corresponds to the arithmetic mean of the molecular weights ofindividual macromolecules. In another embodiment, the dextran used toform the dextran polymer derivative has a molecular weight of from 1,000to 70,000 daltons. In still another embodiment, the dextran used to formthe dextran polymer derivative has a molecular weight of from 1,000 to20,000 daltons. The resulting cationic dextran polymer derivative mayhave a molecular weight ranging from 1,400 to 200,000 daltons, from1,400 to 70,000 daltons, or even from 1,400 to 25,000 daltons.

The cationic dextran polymer derivative comprises an ester-linkedamine-containing substituent. The ester-linked amine-containingsubstituent is selected from

and mixtures thereof, wherein R₁ is selected from C₁, C₂, C₃, or C₄alkyl groups, R₂ and R₃ may be the same or different and are selectedfrom hydrogen, methyl and ethyl groups, and R₄, R₅, and R₆ may be thesame or different and are selected from methyl and ethyl groups.

In one embodiment, the ester-linked amine-containing substituent is

wherein R₁ is selected from C₁, C₂, C₃, or C₄ alkyl groups, and R₂ andR₃ may be the same or different and are selected from hydrogen, methyland ethyl groups. It is to be understood that while the aboveester-linked amine-containing substituent is shown to be in anon-ionized form, the above substituent is meant to include any ionizedforms and salt forms, as one skilled in the art would understand. In oneembodiment, R₁ is a C₃ alkyl group, and R₄, R₅, and R₆ are methylgroups.

In another embodiment, the ester-linked amine-containing substituent is

wherein R₁ is selected from C₁, C₂, C₃, or C₄ alkyl groups, and R₄, R₅,and R₆ may be the same or different and are selected from methyl andethyl groups. In one embodiment, R₁ is a C₃ alkyl group, and R₄, R₅, andR₆ are methyl groups.

In one embodiment, the ester-linked amine-containing substituent ischaracterized by a dissociation constant, known as the pKa. If the pH ofa solution containing the ester-linked amine-containing substituent isthe same as the pKa value, then 50 mol % of the ester-linkedamine-containing substituents are ionized, and 50 mol % are non-ionized.As the pH decreases, a greater fraction of the ester-linkedamine-containing substituents become ionized; as the pH increases, agreater fraction of the ester-linked amine-containing substituentsbecome non-ionized.

In any or all of the above embodiments, the degree of substitution ofthe ester-linked amine-containing substituent may be at least 0.03. Asused herein, the term “degree of substitution” refers to the averagenumber of substituents attached to one repeat, or monomer, of thedextran backbone; the maximum number of ester-linked amine-containingsubstituents that can be attached to a dextran backbone monomer is 3.0.In another embodiment, the degree of substitution of the ester-linkedamine-containing substituents may be at least 0.05. In otherembodiments, higher degrees of substitution are desired. Thus, thedegree of substitution of the ester-linked amine-containing substituentsmay be at least 0.10, at least 0.15, at least 0.2, or even higher.

In another embodiment, the degree of substitution of the ester-linkedamine-containing substituents ranges from 0.03 to 2.95. In yet anotherembodiment, the degree of substitution of the ester-linkedamine-containing substituents ranges from 0.05 to 2.95. Other degrees ofsubstitution of the ester-linked amine-containing substituents may bebeneficial, including from 0.05 to 2.95, from 0.1 to 2.5, from 0.15 to2.0, and even from 0.2 to 2.0.

The cationic dextran polymer derivative further comprises an alkyl estersubstituent selected from acetate, propionate, butyrate, isobutyrate,and mixtures thereof. In another embodiment, the alkyl ester substituentis selected from acetate and propionate. In yet another embodiment, thealkyl ester substituent is acetate. In another embodiment, the alkylester substituent is propionate.

The degree of substitution of the alkyl ester substituent may be atleast 0.05. In another embodiment, the degree of substitution of thealkyl ester substituent may be at least 0.1. In still anotherembodiment, the degree of substitution of the alkyl ester substituentmay be at least 0.5. In yet another embodiment, the degree ofsubstitution of the alkyl ester substituent may be at least 1.0. Inanother embodiment, the degree of substitution of the alkyl estersubstituent ranges from 0.05 to 2.95. In another embodiment, the degreeof substitution of the alkyl ester substituent ranges from 0.1 to 2.5.

The degree of substitution of the substituents may be chosen such thatthe polymer has the desired physical properties. In one embodiment, thedegree of substitution is adjusted to obtain a cationic dextran polymerderivative with the desired aqueous solubility or dispersability. A testto determine the aqueous solubility of a cationic dextran polymerderivative may be performed as follows. The cationic dextran polymerderivative is initially present in bulk powder form with an averageparticle size of greater than 1 micron. The polymer alone isadministered at a concentration of 0.2 mg/mL to a buffer solution at thedesired pH and stirred for approximately 1 hour at room temperature.Next, a nylon 0.45 μm filter is weighed, and the solution is filtered.The filter is then dried overnight at 40° C., and weighed the next day.The aqueous solubility of the polymer is calculated from the amount ofpolymer added to the buffer solution minus the amount of polymerremaining on the filter.

Similar procedures can be used to determine the effect of pH on theaqueous solubility of the cationic dextran polymer derivatives. In thiscase the procedures are performed using aqueous buffer solutions withvarious pH values.

As used herein, by “aqueous soluble” is meant that the cationic dextranpolymer derivative has an aqueous solubility of at least 1 mg/mL in agiven aqueous solution over the pH range specified for the aqueoussolution. By “poorly aqueous soluble” is meant that the polymer has asolubility of less than 0.1 mg/mL over the pH range specified. Factorsaffecting the aqueous solubility of the cationic dextran polymerderivative include the alkyl ester substituent and its degree ofsubstitution, the ester-linked amine-containing substituent and itsdegree of substitution, the pKa of the ester-linked amine-containingsubstituent, the ratio of the degree of substitution of the alkyl estersubstituent to the degree of substitution of the ester-linkedamine-containing substituent, and/or the pH of the aqueous solution.

In one embodiment, the cationic dextran polymer derivative is poorlyaqueous soluble at a pH that is equal to or greater than the pKa valueof the ester-linked amine-containing substituent, but aqueous soluble ata pH less than the pKa value minus 1.

In another embodiment, the degree of substitution of the alkyl estersubstituent is sufficiently high such that when the ester-linkedamine-containing substituent is non-ionized or substantiallynon-ionized, the cationic dextran polymer derivative is poorly aqueoussoluble.

In another embodiment, the cationic dextran polymer derivative isbiocompatible. By “biocompatible” is meant that for some deliveryroutes, the polymer is compatible with and has no significant toxiceffect on the living organism to which it is administered. In oneembodiment, the polymer does not significantly elicit humoral orcell-based immune responses when administered in vivo.

In yet another embodiment, the cationic dextran polymer derivative isbiodegradable. By “biodegradable” is meant that the polymer will degradewhen administered in vivo. By “degrade” is meant that in an in vivo useenvironment, the polymer is broken down into smaller species that can beabsorbed, metabolized, and/or otherwise eliminated or “cleared” from theuse environment. In one embodiment, the polymer degrades within a timeperiod of several days to several weeks. In another embodiment, thepolymer degrades within a time period of several days to several months.In yet another embodiment, the polymer degrades with a time period ofseveral weeks to several months. This degradation can occur throughenzymatic, hydrolytic, oxidative, or other reactions or processes, asare well known in the art. The polymer may also degrade into aqueoussoluble species that can be cleared from the in vivo use environment.For example, the degradation products may be renally cleared through thekidneys or may enter the lymphatic system and then exit through thegastro-intestinal tract.

Synthesis of Cationic Dextran Polymer Derivatives

Cationic dextran polymer derivatives may be synthesized using proceduresknown in the art. In one embodiment, the ester-linked amine-containingsubstituent is first attached to the dextran polymer, and then the alkylester substituent is attached. In another embodiment, the alkyl estersubstituent is first attached to the dextran polymer and then theester-linked amine-containing substituent is attached to the dextranpolymer. In still another embodiment, the ester-linked amine-containingsubstituent and the alkyl ester substituent are simultaneously attachedto the dextran polymer. One skilled in the art will appreciate thatadjustments to the synthetic procedures may be made depending on thesubstituents being attached and the reactivity of the varioussubstituents.

In one embodiment, the cationic dextran polymer derivatives aresynthesized using a homogeneous reaction by first dissolving the dextranpolymer in a suitable solvent. Suitable solvents include, but are notlimited to, dimethylformamide (DMF), dimethylacetamide (DMAC),formamide, dimethylsulfoxide (DMSO), methylene chloride, and mixturesthereof. Reactants and any catalysts and/or co-reactants are added tothe reaction mixture, and the mixture is allowed to react at anappropriate temperature and for an appropriate time to achieve thedesired degree of substitution. The reaction mixture may then bequenched, and the derivatized polymer precipitated and washed. Thederivatized polymer may be purified prior to use or prior to furtherreaction. One skilled in the art will understand that standard polymerderivatization techniques may be applied to the synthesis of cationicdextran polymer derivatives. See for example Advances in PolymerScience, 205, Polysaccharides II, edited by Dieter Klemm(Springer-Verlag, Berlin Heidelberg, 2006). The specific reactionconditions used to attach the ester-linked amine-containing substituentsand alkyl ester substituents will vary depending on the properties ofthe substituent. In addition, for some reactants, protecting groups maybe added to the reactants, and after performing the reaction, theprotecting groups may be removed to form the desired substituent.

When amine-containing substituents are ester linked to dextran,activation of the carboxylic acid and/or the use of coupling agents isoften utilized to increase the rate of reaction and improve yield.Activation or coupling agents such as N,N′-carbonyldiimidazole (CDI) andN,N′-dicyclohexylcarbodiimide (DCC) may be employed using techniquesknown in the art. Similar reactions can be obtained usingamine-containing substituents based on carboxylic acid chlorides andanhydrides. Such reactions often utilize the presence of a base tocatalyze the reaction. See for example, T. Heinze, et al., Advances inPolymer Science, Vol. 205, pp. 199-291, 2006. A similar reaction schemecan be used to attach alkyl ester substituents to the dextran polymer.

Other features and embodiments of the disclosure will become apparentfrom the following Examples that are given for illustrating theinvention rather than for limiting its intended scope.

EXAMPLES

Polymer 1, dextran propionate quaternary amine having the structure anddegree of substitution shown in Table 1, was synthesized using thefollowing procedure. First, 3.53 g (3-carboxypropyl)trimethyl ammoniumchloride was dissolved in 30 mL of a solvent consisting of 1:2 (vol:vol)dimethyl formamide (DMF)/formamide. To this solution was added 3.52 g1,1-carbonyldiimidazole with rapid stirring. Next, a second solution wasformed by dissolving 10.01 g dextran (that had been dried for 24 hoursat 90° C.) in 50 mL formamide at 50° C. The dextran had an averagemolecular weight of 40,000 daltons (available from Amersham Biosciences,Piscataway, N.J.). After the first solution had stirred for 3.5 hours,the two solutions were combined and stirred 18.5 hours at 50° C. in asealed round bottom flask equipped with a condenser.

To form the dextran propionate quaternary amine, 8.89 g sodiumpropionate and 50 mL formamide was added to the reaction mixture, andthe mixture stirred at 50° C. for about an hour. To this, 23.06 gpropionic anhydride was added, and stirring continued at 50° C. forabout 2 hours before an additional 9.41 g propionic anhydride was added.Stirring continued at 50° C. for 18 hours following addition of thesecond aliquot of propionic anhydride.

Approximately half of the reaction mixture was poured into 800 mL water,and sodium chloride was added until the solution became cloudy. Thecloudy gelatinous mixture was centrifuged to isolate the solid polymer.The solid polymer was collected and added to 600 mL water, and theslurry was centrifuged to isolate solid product. This water wash processwas repeated. The solid product was dissolved in 350 mL methanol. Tothis, 400 mL isopropyl alcohol (IPA) was added and the solvent wasremoved using rotary evaporation under vacuum (rotoevaporated). Anadditional 200 mL IPA was added, followed by solvent removal. The solidpolymer was dried under vacuum for 48 hours. For a final purificationstep, the polymer was dissolved in methylene chloride, filtered througha 0.2 μm filter, and rotoevaporated. The solid polymer was dried in avacuum desiccator.

The polymer was analyzed by ¹³C nuclear magnetic resonance (NMR)spectroscopy to examine propionate and quaternary amine substitutions onthe dextran backbone (see FIG. 1). For NMR analysis, polymer sampleswere dissolved in deuterated dimethyl sulfoxide (DMSO) at aconcentration of 200 mg/mL. Propionate and quaternary amine groupconcentrations were determined using the ratios of peak areas to thepeak area of the anomeric carbon in the dextran ring. Polymer propertiesare shown in Table 1.

TABLE 1 Starting Degree of Dextran/ Degree of Substitution of MolecularSubstitution of Ester-Linked the Amine- Weight Alkyl Ester the AlkylEster Amine-Containing Containing Polymer (Daltons) SubstituentSubstituent Substituent Substituent 1 Dextran 40,000 propionate 2.8

0.22 2 Dextran 40,000 acetate 1.0

0.14 3 Dextran 20,000 acetate 1.9

0.14 4 Dextran 10,000 propionate 1.9

0.04 5 Dextran 20,000 propionate ND*

ND *ND = not determined

Polymer 2, dextran acetate quaternary amine having the structure anddegree of substitution shown in Table 1, was synthesized using thefollowing procedure. First, dextran quaternary amine was synthesized bydissolving 3.005 g (3-carboxypropyl) trimethylammonium chloride in 30 mLDMF/formamide (1:2 vol:vol). To this, 3.004 g 1,1-carbonyldiimidazolewas added in small portions (200-300 mg) with rapid stirring. Afterfoaming ceased, the solution was stirred for 2 hours at roomtemperature. Next, a second solution was formed by dissolving 9.023 gdextran having an average molecular weight of 40,000 daltons (AmershamBiosciences; dried at least 24 hours at 90° C.) in 50 mL formamide at50° C. The two solutions were combined and stirred for about 18 hours at50° C. in a sealed round bottom flask equipped with a condenser.

To form the dextran acetate quaternary amine, 8.779 g sodium acetate wasadded to the reaction mixture, followed by 7.144 g acetic anhydride. Thereaction mixture was stirred at 50° C. for 5 hours. The reaction mixturewas removed from the heat and stirred about 18 hours at roomtemperature.

Approximately one third of the reaction mixture was poured into 800 mLacetone to precipitate the polymer and remove organic solvent. The solidpolymer material was collected by filtration (Whatman 113 filter paper,Piscataway, N.J.), added to 400 mL acetone, and filtered again. Thefiltrate material was rinsed with 200 mL acetone and dried under vacuum.For a final purification step, the polymer was dissolved in 150 mL waterand the pH was adjusted to about 5 using 6N HCl. The polymer solutionwas poured into a dialysis membrane tube (6000 to 8000 dalton molecularweight cut off dialysis membrane) and dialyzed for about 2 days usingdeionized water. Fresh water was added (3.5 L) and dialysis continuedfor an additional day. After dialysis, IPA was added to the polymersolution (3:1), and the solution was rotoevaporated to dryness. Thesolid polymer was dried under vacuum. The ¹³C NMR spectrum of Polymer 2is shown in FIG. 2.

Polymer 3, dextran acetate quaternary amine having the structure anddegree of substitution shown in Table 1, was synthesized using thefollowing procedure. First, Texas Red dye was attached to dextran forpolymer detection. To attach the dye, 6.145 g of dextran having anaverage molecular weight of 20,000 daltons (Amersham Biosciences; dried3 days at 90° C.) was dissolved in 50 mL formamide at 50° C., and 1.495g diisopropylethylamine was added to the reaction mixture. Next, 35 mgof Texas Red dichlorotriazine (available from Invitrogen Corp.,Carlsbad, Calif.) was added and rinsed into the dextran solution usinganhydrous DMF (20 mL total). The reaction mixture was stirred overnightat 50° C.

Dextran quaternary amine was synthesized by dissolving 1,350 g(3-carboxypropyl) trimethylammonium chloride and 1.479 g1,1-carbonyldiimidazole in 50 mL DMF/formamide (1:1) at roomtemperature. This solution was stirred for 2 hours, then added to thedextran-dye reaction mixture above. The reaction was stirred at 50° C.for about 18 hours. The polymer was isolated by precipitation into 900mL IPA followed by filtration. Next, the polymer was dissolved in 150 mLformamide, precipitated by adding 75 mL of the polymer solution to 900mL of IPA/ethyl acetate (1:1), and filtered (Whatman 113 filter paper).The product was rinsed with several hundred mL IPA and finally about 200mL diethyl ether. The solid polymer was dried under vacuum.

To form the dextran-dye acetate quaternary amine, 4.146 g dextran-dyequaternary amine was dissolved in 50 mL formamide at 50° C., and 5.124 gsodium acetate was added to the reaction mixture, followed by 7.073 gacetic anhydride. The reaction mixture was stirred at 50° C. for about18 hours.

The reaction mixture was quenched in 400 mL rapidly-stirred watersaturated with sodium chloride, forming a gelatinous precipitate. Thegelatinous mixture was centrifuged to isolate the solid polymer. Thesolid polymer was collected and added to 300 mL water, and the slurrywas centrifuged to isolate solid product. This water wash process wasrepeated. The solid product was dissolved in 150 mL methanol, andfiltered using a 20 μm filter. To this, 100 mL IPA was added and thesolution was rotoevaporated to dryness. The solid polymer was driedunder vacuum. The ¹³C NMR spectrum of Polymer 3 is shown in FIG. 3.

Polymer 4, dextran propionate quaternary amine having the structure anddegree of substitution shown in Table 1, was synthesized using thefollowing procedure. First, dextran propionate was made by dissolving30.057 g dextran having an average molecular weight of 10,000 daltons(available from Amersham) in 100 mL formamide at 50° C. To this, 10.778g sodium propionate and 65.141 g propionic anhydride was added, andstirring continued at 50° C. for about 18 hours. The resulting dextranpropionate was precipitated into 800 mL water in a blender and filteredthree times (Whatman 113 filter paper), and the filtrate was washed withan additional 500 mL water. This material was air-dried overnight.

Dextran propionate quaternary amine was synthesized by dissolving 0.661g (3-carboxypropyl) trimethylammonium chloride and 0.665 g1,1-carbonyldiimidazole in 50 mL formamide. The solution was stirred for6.5 hours. To this, 4.015 g dextran propionate (above) was added, andthe reaction mixture was stirred for about 16 hours.

The reaction mixture was poured into 800 mL water, and sodium chloridewas added until the material flocculated. The material was filteredusing a 0.45 μm filter to isolate the solid polymer. The material wasdissolved in methanol, and filtered (Whatman 113 filter paper). Thesolution was rotoevaporated to dryness. The polymer was dried undervacuum for 3 hours, dissolved in 80 mL methanol, and precipitated into400 mL water with stirring. The polymer suspension was poured into adialysis membrane tube (6000 to 8000 dalton molecular weight cut offdialysis membrane) and dialyzed using deionized water. Followingdialysis, the suspension was mixed with IPA (7:3 IPA/polymersuspension), and rotoevaporated to dryness. The solid polymer wasdissolved in 50 mL methanol and rotoevaporated to dryness again. Thepolymer was dried under vacuum. The ¹³C NMR spectrum of Polymer 4 isshown in FIG. 4.

Polymer 5 dextran propionate primary amine, having the structure shownin Table 1, was synthesized using the following procedure. First,dextran primary amine was synthesized by dissolving 5.027 gN-(9-Fluorenylmethoxycarbonyl)-beta-alanine (Fmoc-β-alanine) and 2.701 g1,1-carbonyldiimidazole in 50 mL dimethylformamide (DMF). Next, a secondsolution was formed by dissolving 7.039 g dextran having an averagemolecular weight of 20,000 daltons (Amersham Biosciences; dried 2 daysat 90° C.) in 50 mL formamide. After the first solution had stirred forseveral hours, the two solutions were combined and stirred overnight atroom temperature in a sealed round bottom flask equipped with acondenser.

To form the dextran propionate primary amine, 9.41 g sodium propionateand 34.62 g propionic anhydride was added to the reaction mixture andheated to about 50° C. until the mixture was clear. The reaction mixturewas stirred overnight at room temperature.

To isolate the product, the reaction mixture was poured into 1800 mLwater with stirring to precipitate the polymer. The solid polymermaterial was collected by filtration (Whatman 113 filter paper), andrinsed with 400 mL water. The material was redissolved in 100 mL acetoneto form a clear solution. The acetone solution was then poured into 1800mL water, and sodium chloride was added to precipitate the polymermaterial. The material was filtered and rinsed with 200 mL water, anddried on filter paper (Whatman 113 filter paper) with the vacuum.

The Fmoc protecting group was removed by adding 6.93 g of dried productto 50 mL DMF. Next, 15 mL piperidine was added, and the mixture wasstirred for 45 minutes at room temperature. The solution was poured into1800 mL water containing 13 mL of 12 N HCl. Sodium chloride was addeduntil saturation was obtained, to precipitate the polymer. The polymerwas collected by filtration (Whatman 113 filter paper) and rinsed withwater. The polymer was redissolved in methanol, the solution wasfiltered through a 5 μm filter, and the solvent was removed using rotaryevaporation under vacuum. The solid polymer was dissolved again inmethanol, filtered, and rotoevaporated to dryness. The material wasfurther purified by adding 1.03 g polymer and 2.1 g piperidine on RastaResin (polystyrene crosslinked with divinylbenzene scavenger resinbeads, available from Sigma-Aldrich Co.) to 20 mL methanol:methylenechloride 1:1, and stirring overnight at room temperature. The RastaResin was removed by filtration (20 μm nylon filter), and the solventwas rotoevaporated to dryness. A ninhydrin test on the polymer spottedon silica confirmed the presence of the primary amine functional group.

In one embodiment, a dextran polymer derivative comprises (a) anester-linked amine-containing substituent, selected from

and mixtures thereof, wherein R₁ is selected from C₁ to C₄ alkyl groups,R₂ and R₃ may be the same or different and are selected from hydrogen,methyl and ethyl groups, and R₄, R₅, and R₆ may be the same or differentand are selected from methyl and ethyl groups, and wherein the degree ofsubstitution of said ester-linked amine-containing substituent is atleast 0.03, and (b) an alkyl ester substituent selected from acetate,propionate, butyrate, isobutyrate, and mixtures thereof, wherein thedegree of substitution of said alkyl ester substituent is at least 0.05.

In some embodiments, the ester-linked amine-containing substituent is

wherein R₁ is selected from C₁ to C₄ alkyl groups, and R₂ and R₃ may bethe same or different and are selected from hydrogen, methyl and ethylgroups. In certain embodiments, R₁ is a C₂ alkyl group, and R₂ and R₃are hydrogens.

In some embodiments, the ester-linked amine-containing substituent is

wherein R₁ is selected from C₁ to C₄ alkyl groups, and R₄, R₅, and R₆may be the same or different and are selected from methyl and ethylgroups. In certain embodiments, R₁ is a C₃ alkyl group, and R₄, R₅, andR₆ are methyl groups.

In any or all of the above embodiments, the ester-linkedamine-containing substituent may have a degree of substitution of atleast 0.05. In some embodiments, the ester-linked amine-containingsubstituent has a degree of substitution of at least 0.1.

In any or all of the above embodiments, the degree of substitution ofthe alkyl ester substituent may be at least 0.1. In any or all of theabove embodiments, the degree of substitution of the alkyl estersubstituent is at least 0.5. In any or all of the above embodiments, thedegree of substitution of the alkyl ester substituent is at least 1.0.

In any or all of the above embodiments, the alkyl ester substituent maybe selected from acetate and propionate. In any or all of the aboveembodiments where R₁ is a C₂ alkyl group, and R₂ and R₃ are hydrogens,the alkyl ester substituent may be propionate. In any or all of theabove embodiments where R₁ is a C₃ alkyl group, and R₄, R₅, and R₆ aremethyl groups, the alkyl ester substituent may be acetate. In any or allof the above embodiments where R₁ is a C₃ alkyl group, and R₄, R₅, andR₆ are methyl groups, the alkyl ester substituent may be propionate.

The terms and expressions which have been employed in the foregoingspecification are used therein as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding equivalents of the features shown and describedor portions thereof, it being recognized that the scope of the inventionis defined and limited only by the claims which follow.

What is claimed is:
 1. A dextran polymer derivative comprising: (a) anester-linked amine-containing substituent, selected from

 and mixtures thereof, wherein R₁ is selected from C₁, C₂, C₃, or C₄alkyl groups, R₂ and R₃ may be the same or different and are selectedfrom hydrogen, methyl and ethyl groups, and R₄, R₅, and R₆ may be thesame or different and are selected from methyl and ethyl groups, andwherein the degree of substitution of said ester-linked amine-containingsubstituent is at least 0.03; and (b) an alkyl ester substituentselected from acetate, propionate, and mixtures thereof, wherein thedegree of substitution of said alkyl ester substituent is at least 0.05.2. The dextran polymer derivative of claim 1 wherein said ester-linkedamine-containing substituent is

wherein R₁ is selected from C₁, C₂, C₃, or C₄ alkyl groups, and R₂ andR₃ may be the same or different and are selected from hydrogen, methyland ethyl groups.
 3. The dextran polymer derivative of claim 2 whereinR₁ is a C₂ alkyl group, and R₂ and R₃ are hydrogens.
 4. The dextranpolymer derivative of claim 3 wherein said alkyl ester substituent ispropionate.
 5. The dextran polymer derivative of claim 1 wherein saidester-linked amine-containing substituent is

wherein R₁ is selected from C₁, C₂, C₃, or C₄ alkyl groups, and R₄, R₅,and R₆ may be the same or different and are selected from methyl andethyl groups.
 6. The dextran polymer derivative of claim 5 wherein R₁ isa C₃ alkyl group, and R₄, R₅, and R₆ are methyl groups.
 7. The dextranpolymer derivative of claim 1 wherein said ester-linked amine-containingsubstituent has a degree of substitution of at least 0.05.
 8. Thedextran polymer derivative of claim 7 wherein said ester-linkedamine-containing substituent has a degree of substitution of at least0.1.
 9. The dextran polymer derivative of claim 1 wherein said degree ofsubstitution of said alkyl ester substituent is at least 0.1.
 10. Thedextran polymer derivative of claim 9 wherein said degree ofsubstitution of said alkyl ester substituent is at least 0.5.
 11. Thedextran polymer derivative of claim 10 wherein said degree ofsubstitution of said alkyl ester substituent is at least 1.0.
 12. Thedextran polymer derivative of claim 1 wherein the polymer has amolecular weight ranging from 1,400 to 200,000 daltons.
 13. The dextranpolymer derivative of claim 12 wherein the polymer has a molecularweight ranging from 1,400 to 25,000 daltons.
 14. The dextran polymerderivative of claim 1 wherein the polymer is biocompatible.
 15. Thedextran polymer derivative of claim 1 wherein the polymer isbiodegradable.
 16. The dextran polymer derivative of claim 1, whereinthe dextran polymer derivative has an aqueous solubility of at least 1mg/mL at a pH less than a pKa value of the ester-linked amine-containingsubstituent minus 1.